Bovine mastitis is one of the most costly and complex diseases of the dairy industry3,9. The complexity is reflected in the numerous causative pathogens, the variety and magnitude of the physiological responses to these pathogens and the variation in efficacy of control measures for different causative organisms4,7,17,18,23,32. Coagulase-negative staphylococci (CNS) have been traditionally considered minor pathogens. However, their importance has increased because they have become the most frequently isolated group of species from bovine milk in many areas around the world16,29,33. CNS usually cause subclinical mastitis (SCM), resulting in an increase in the somatic cell count (SCC) and a decrease in milk quality with the resulting economic losses that this implies30. The understanding and control of CNS mastitis are complicated by the heterogeneity of this group of bacteria. So far, based on Jean Euzéby's List of Procaryotic names with Standing in Nomenclature (http://www.bacterio.net/s/staphylococcus.html), the genus Staphylococcus contains 47 species and 24 subspecies. Even though, the prevalence of CNS species varies among the studies; six species have been reported to be commonly involved in intramammary infections (IMI)20,30. Recently, it has been found that CNS speciation based on the biochemical profile is not accurate enough for bovine CNS identification26. Hence, molecular methods have become an important diagnostic tool to improve CNS species differentiation5,15,39. CNS tend to be more resistant to antibiotics than Staphylococcus aureus and easily develop multiresistance2,27,32. In addition, limited information is available regarding differences in antimicrobial susceptibility among CNS species identified by genotyping27,38. As yet, nothing has been reported about the prevalence and susceptibility antibiotics of CNS species of dairy cows in Argentina. Recently, a research conducted in our laboratory8 showed that CNS represent the most prevalent bacterial group of minor pathogens isolated from the subclinical infection of dairy cows in Córdoba province, Argentina. The aim of the present study was to determine the prevalence and antibiotic resistance profiles of CNS species isolated from bovine subclinical mastitis in 51 dairy herds in the central region of Argentina.

Genotypic methods have shown to have higher specificity and sensitivity39 than other methods for discriminating among species, resulting in a better alternative for the identification of CNS isolates. A number of PCR amplicon sequencing-based methods for the identification of CNS have been reported5,26,30. Amplified fragment length polymorphism (AFLP)20,31 and PCR-restriction fragment length polymorphism (RFLP) applied to the groEL or gap genes have been used to identify CNS isolated from bovine milk15,25. The discrepancy observed in the distribution of CNS species identified by genotyping causing SCM in different countries, might be due to differences in experimental design10,20,30,31,35,38 however, S. chromogenes was one of the most prevalent species both in this work and in previous studies carried out in other countries such as Finland, Sweden, Belgium, Canada and Brazil, ranging between 23.3% and 78.8% of CNS isolates. To our knowledge, only Piessens et al.20 revealed that S. haemolyticus was the second most prevalent CNS species as in our study causing 27.6% of cases. Special attention should be given to both species, considering that the udder was found to be their main reservoir20.

In our study, a minimum of three different species of CNS were present in 41.2% of the herds. S. chromogenes and S. haemolyticus were isolated in most of the farms. This is in line with other studies, where these species have been isolated from all analyzed herds20,30. Although the reasons for the increased prevalence of some CNS species are not known, these might be attributed to the ability of each species to adapt to the mammary gland10,20 or to the different management patterns of mastitis control schemes30.

One limitation to the present study is the fact that we cannot be sure that the CNS were isolated from the diseased quarter since other non-infectious factors could cause an inflammatory response. However, CNS in pure culture allow to ensure that they are the only etiological agents causing subclinical mastitis.

The direct comparison of studies on antimicrobial susceptibility of CNS is often difficult mainly because of the use of different methodologies and breakpoints for testing susceptibility. A great variability has been reported in the MIC90 values for penicillin in CNS, ranging from 0.12μg/ml to 32μg/ml2,11,12,19,22,24,34. The MIC90 value for penicillin found in our study was well above the recommended breakpoint (CLSI, 2008). Moreover, the penicillin resistance percentage was higher than that described by Gentilini et al.11 in Argentina and those reported in other countries such as Uruguay12, The Netherlands27, USA22 and Sweden2. In the last decade, Gentilini et al.11 reported methicillin resistance among CNS isolated from bovine mastitis in our country. Since continuous surveillance is needed for the early detection of this kind of resistance, oxacillin has been used in this study to test susceptibility to methicillin. The MIC90 value for oxacillin was similar to those reported by different authors11–13,24,28. The percentage of resistant strains found was higher than that reported by other authors2,11–13, and similar to that reported in studies from Finland, Germany, USA, Sweden and The Netherlands19,21,22,27,28. The variability in the MIC90 values of the antimicrobial activity of penicillin and oxacillin against CNS highlights the relevance of determining β-lactams susceptibility patterns for this bacterial group. Macrolides are frequently used in Argentina for bovine mastitis treatment, since high concentrations in milk are obtained following parenteral administration. Although erythromycin was evaluated as representative of this group, tylosin is used in the therapy. Tetracycline was included in this study for epidemiological purposes as representative of tetracyclines; however, they are not recommended for mastitis treatment in Argentina because of the risk of an extended residue presence in milk. The MIC90 value for erythromycin and tetracycline was higher than that observed by several authors in Argentina, Finland, Germany, Sweden and USA2,11,13,19,21,28. Although clindamycin is not used in the treatment of bovine mastitis, the determination of the MIC for this antibiotic was conducted in order to test susceptibility to lincomycin; however, specific interpretative breakpoints are defined for pirlimicyn6. Susceptibility to pirlimycin could not be inferred from clindamycin results because some mechanism (enzymatic modification) does not exhibit cross-resistance. The MIC90 value for clindamycin obtained in this study was similar to that previously described by Gentilini et al.11 in our country and higher than that reported by other investigators in different countries2,13,19,21. The proportion of CNS resistant to a single compound or two related compounds obtained in our study was found to be similar to that described by Sampimon et al.27S. haemolyticus and S. chromogenes were the most frequent resistant species; therefore, it would also be hypothesized that they should be more “at risk” of developing antibiotic resistance or acquiring resistance determinants. Conversely, the absence or lower percentages of resistant isolates to multiple drugs for both species were found by Sampimon et al.27 We conclude that CNS species from bovine subclinical mastitis differ in prevalence and antimicrobial resistance profiles, which may have implications for treatment and management decisions when CNS are the predominant bacterial group on dairy farms.

The authors declare that they have no conflicts of interest.